(520) Molecular evidence for Mycobacterium abscessus in NTM patient cohorts classified by sputum culture

Background: M. abscessus (MAB) is an important CF pathogen, and culture is the only test utilized for detection and assessment of treatment response. However, sputum culture has low sensitivity due to required decontamination procedures in people with CF (pwCF). Additionally, the adoption of highly effective modulator treatments in pwCF has reduced the availability of sputum samples and the culture sensitivity in pwCF known to have infection, thus increasing the need for culture independent detection from minimal sample volumes. We hypothesized that a species-specific molecular amplification assay could detect MAB DNA from total sputum DNA.

Methods: A TaqMan real-time quantitative PCR (qPCR) assay was optimized for detection of MAB DNA. Total DNA was extracted from 100uL of raw sputum homogenized in PBS. The MAB qPCR assay was evaluated in total DNA from 120 sputum samples from 86 pwCF enrolled in the PREDICT and PATIENCE trials with a history of NTM. Twenty-nine subjects had paired samples from more than one date. Ninety samples were linked to NTM culture results from the same sample, and 99 had culture results within 30 days. A cumulative NTM culture history from all available AFB cultures was collected for each subject, and 45% had cultured positive for MAB on at least one occasion. MAB DNA abundance for each sample was extrapolated from a standard curve ranging from 10pg/uL to 0.001pg/uL MAB DNA.

Results: Of 120 samples, 24 (20%) detected MAB DNA by qPCR. When compared to NTM culture results from the same sample (n=90), the sensitivity and specificity of molecular MAB detection versus culture was 0.79 and 0.91, respectively, with a positive predictive value (PPV) of 61% and a negative predictive value (NPV) of 96%. When compared to culture results within 30 days (n=99), the sensitivity and specificity of molecular MAB detection versus culture was 0.78 and 0.90, respectively, with a PPV of 64% and an NPV of 95%. The average molecular burden of MAB DNA was 0.08 pg/uL (range: 0.002 – 0.58 pg/uL) in culture positive samples, and 0.07pg/uL (range: 0.003 – 0.53 pg/uL) in a subset of 10 culture negative samples. Among the 10 samples that were molecular MAB positive and culture negative, 5 were from subjects who had prior MAB positive cultures ranging from 62 – 1402 days before sample collection, and 5 were from subjects with no history of positive MAB cultures. Among subjects with >1 sample (n=51), molecular detection in either had a sensitivity and specificity of 0.88 and 0.65, for a positive culture within 30 days.

Conclusions: Molecular amplification by qPCR using low inputs of total sputum DNA is a sensitive and specific method for detection of MAB compared to traditional airway culture. In some cases, qPCR may detect MAB that is nonculturable with current decontamination methods. Analysis of >1 samples may improve the sensitivity of the assay.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (NICK17K0, NICK20Y2OUT) and the NIH (R01HL146228).

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